1. Field of the Invention
The invention relates to a universal method for isolating nucleic acids from very different starting materials containing nucleic acids, wherein the method guarantees a very high quality of the isolated nucleic acids as well as allows the isolation of quantitative yields.
2. Discussion of the Background
Under conventional conditions, the isolation of DNA from cells and tissues is carried out such that the starting materials containing the nucleic acids are digested under highly denaturing and reducing conditions with, in part, the use of protein-degrading enzymes, the released nucleic acid fraction is then purified in phenol/chloroform extraction stages and the nucleic acids are isolated by dialysis or ethanol precipitation from the aqueous phase (Sambrook, J., Fritsch, E. F. und Maniatis, T., 1989, CSH, “Molecular Cloning”).
These conventional methods for the isolation of nucleic acids from cells and especially from tissues are very time consuming (in part longer than 48 h), require considerable apparative expenditure and moreover are not realizable under field conditions. In addition such methods are hazardous to health owing to the chemicals used in amounts that are not inconsiderable, such as phenol and chloroform.
Different alternative methods for the isolation of nucleic acids from different biological starting materials allow the elaborate and health-damaging phenol/chloroform extraction of nucleic acids to be circumvented and a reduction in time expenditure to be achieved.
All of these methods are based on a method for the preparative and analytical purification of DNA fragments from agarose gels developed and described for the first time by Vogelstein and Gillespie (Proc. Natl. Acad. Sci. USA, 1979, 76, 615-619). The method combines the dissolution in a saturated solution of a chaotropic salt (NaI) of the agarose containing the bands of the DNA to be isolated with binding of the DNA to glass particles. The DNA fixed to the glass particles is then washed with a wash solution (20 mM Tris HCl [pH 7.2]; 200 mM NaCl; 2 mM EDTA; 50% v/v ethanol) and then separated from the support particles.
Until now this method has undergone a series of modifications and is currently used for different methods for the extraction and purification of nucleic acids from different sources (Marko, M. A., Chipperfield, R. und Birnboim, H. G., 1982, Anal. Biochem., 121, 382-387).
In addition, a multiplicity of reagent systems exists world-wide today, predominantly for the purification of DNA fragments from agarose gels and for the isolation of plasmid DNA from bacterial lysates, and also for the isolation of longer chain nucleic acids (genomic DNA, cellular total RNA) from blood, tissues or also cell cultures.
All these commercially available kits are based on the well-known principle of binding nucleic acids to mineral supports in the presence of solutions of different chaotropic salts, and use suspensions of finely-milled glass powder (e.g. Glasmilk, BIO 101, La Jolla, Calif.), diatomaceous earths (Sigma company) or silica gels as support materials.
A method for the isolation of nucleic acids is illustrated which is practicable for a number of different applications proposed in U.S. Pat. No. 5,234,809 (Boom). A method is described therein for the isolation of nucleic acids from starting materials containing nucleic acids, whereby the starting material is incubated with a chaotropic buffer and a DNA-binding solid phase. The chaotropic buffer carries out both the lysis of the starting material as well as the binding of the nucleic acids to the solid phase. The method is well suited for the isolation of nucleic acids from small amounts of sample and finds practical use particularly in the area of the isolation of viral nucleic acids.
Specific modifications of these methods concern the use of novel support materials which have applicative advantages for particular problems (WO-A 95/34569).
More recent patent applications disclose that so-called anti-chaotropic salts can be used very efficiently and successfully as components of lysis/binding buffer systems for the adsorption of nucleic acids to silicate materials known and used by the person skilled in the art (EP 1135479). The advantage of this method is that by circumvention of the use of chaotropic salts a clearly lower hazard to health is posed by the extraction system. However, on the other hand, high salt concentrations (>1.5 M) are required in the lysis buffer for an efficient isolation of nucleic acids from a complex biological sample especially with respect to a highest possible nucleic acid recovery. Thus, the document discloses that the lysis buffers used contain salt concentrations of 1.5 M-3 M.
A method is described in DE 4321904 in which an efficient isolation of nucleic acids is possible with a combination of chaotropic high salt buffer in with alcoholic components. The lysis buffers disclosed in DE 4321904 thereby always contain salt concentrations of 4 M-8 M; guanidine hydrochloride, guanidine thiocyanate or potassium iodide in particular are used as salts. It is known that these salts bring about lysis of the starting material as well as potent inactivation of nucleolytic enzymes. The addition of an alcohol is carried out after lysis of the starting material. The patent discloses that the addition of the alcoholic component to the high salt lysis buffer mediates a highly efficient binding of the nucleic acids to the silicate filter material employed. The disadvantage of the use of lysis buffers with high ion strength chaotropic salts is, however, always the restricted and also inefficient use of additional proteolytic enzymes for an effective digestion of complex biological samples, for these enzymes are themselves damaged by the protein-denaturing action of chaotropic buffers. Furthermore, extensive wash stages are needed subsequently to remove the high salt concentrations from the adsorption material employed. It is known to the person skilled in the art that chaotropic salts exert a high inhibitory action on a number of down-stream applications.
Analysis of the state of the art points out quite impressively that a plurality of possibilities exists for binding nucleic acids to solid support materials, in particular silicon-based mineral support materials, then to wash and to release once more the nucleic acids from the support material. It thereby becomes very clear that so-called chaotropic salts or so-called anti-chaotropic salts are added for the isolation of nucleic acids from complex biological samples.
Chaotropic components are a substances that destroy regular structures of liquid water based on the formation of hydrogen bonds, in that they inhibit the formation of H2O cage structures necessary for solvation. Examples of chaotropic components are thiocyanates, iodides or perchlorates.
Anti-chaotropic components are substances that enhance regular structures of liquid water based on the formation of hydrogen bonds. Examples of anti-chaotropic components are ammonium, sodium or potassium salts.
Non-chaotropic components are, for example salts, that are between chaotropic and anti-chaotropic salts, and include for example, magnesium chloride or aluminium chloride. Non-chaotropic compounds do not enhance or destroy regular structures of liquid water based on the formation of hydrogen bonds. Non-chaotropic substances are, for example, those in the middle of the Hofmeister series of salts.
The advantages of the use of chaotropic salts for processes for the isolation and purification of nucleic acids are founded in the fact that these salts and buffers derived from them effect an efficient denaturing of proteins. If necessary, this makes possible the isolation of nucleic acids even from complex biological samples, without the use of proteolytic enzymes. A further advantage consists in the fact that chaotropic salts, as components of lysis buffers, also effect a potent inactivation of RNases, in particular in the isolation of RNA. It is, however, disadvantageous that the use of DNA and RNA extraction procedures on the basis of chaotropic salts is always bound to high ion strengths. In low concentrations, an efficient bonding of nucleic acids to chromatographic materials used until now is not possible. This has the result, among others, that washing steps known to those skilled in the art are very complex, founded in separating the high salt concentrations from the final DNA or RNA to be isolated. It is also known that chaotropic components exert a high inhibitory action on a number of down-stream applications. What is more, chaotropic salts are hazardous to health and, in the end, also very cost-intensive.
Advantages of the input of so-called anti-chaotropic salts or non-chaotropic salts (in the patent application cited, this means the groups of the salts which stand at the other end of the Hofmeister Series in relation to the chaotropic salts) consist in the fact that buffer formulations derived from these salts can also be employed for the isolation of nucleic acids. However, as these salts are so-called protein-stabilizing salts, the digestion of complex biological samples always takes place only in the presence of proteolytic enzymes. The efficiency of lysis processes is thereby, as a rule, always worse than with the use of chaotropic salts as a component of lysis buffers. What is more, it is disadvantageous that, particularly in the isolation of RNA from complex biological samples, no inactivation of RNases takes place. This has the result that an efficient isolation of RNA by means of non-chaotropic buffer systems is not possible. It is shown in the analysis of the background art to date that ion strengths of >1M must be introduced for anti-chaotropic methods for the isolation of nucleic acids as well, in order to achieve an efficient and quantitative isolation of nucleic acids.